Phylotype IIB Sequevar Research Articles

Differentiation of Cool-Virulent Strains in the Ralstonia solanacearum Species Complex by Melt Curve of a DNA Fragment from Effector Gene Rips1

The Ralstonia solanacearum species complex ( Rssc) causes bacterial wilt diseases across hundreds of plant species. Phylogenetic analysis of DNA sequences has allowed the grouping of Rssc strains into phylotypes based on genetic relatedness. Although specific phenotypes are associated with corresponding phylotypes, the molecular bases for these differences are not always well understood. The complex trait of cool-virulence that allows several members of phylotype II to cause disease at temperatures below 25°C is one of these poorly understood traits. Here, we report that a 96-bp DNA fragment from a gene encoding the type III secretion system effector protein RipS1, which was recently reported to play a role in the cool-virulence phenotype, has a melting temperature that varies based on the strain of origin. By testing 81 Rssc strains with melt-curve analysis, we found a bimodal distribution with clear separation between the group of strains with a melting temperature below 87.5°C that includes phylotypes I, III, and IV strains and the group with a melting temperature above 87.5°C that includes all 35 tested select agent race 3 biovar 2 (phylotype IIB sequevars 1 and 2) and seven phylotype IIB sequevar 4 strains, previously known to possess cool-virulence capability, with a few exceptions. This provides further support that sequence differences in this effector differentiate cool-virulent strains from non-cool-virulent strains and show potential for use as a marker in continued studies of the cool-virulence phenotype. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.

First report of Ralstonia solanacearum causing bacterial wilt of eucalyptus in Ecuador.

In January 2018, eucalyptus trees showing wilt symptoms were identified in a commercial plantation located in the Quevedo Cantón, Los Ríos province, Ecuador. Disease incidence reached 40% of the eucalyptus field and affected plants displayed reddening and wilting of the foliage, leaf drop, branch dieback, and reduced growth, resembling bacterial wilting symptoms (Santiago et al., 2014). Transverse sections made on trunks of wilted trees revealed vascular discoloration of the wood and bacterial ooze. Wood pieces taken from discoloured tissues were surface disinfected and macerated in sterilized distilled water. The resulting suspensions were spread on plates containing triphenyltetrazolium chloride medium (TZC) (Kelman, 1954) and incubated at 28°C for 48 h. Three mucoid white colonies with pinkish centers, characteristic of Ralstonia solanacearum, were purified (Figure S1C). The isolates were Gram negative in the KOH test, non-fluorescent on King's B medium, and formed cream colonies on yeat extract-dextrose-calcium carbonate (YDC) medium. Phylotype-specific multiplex PCR (PMX-PCR) (Fegan and Prior, 2005) and phylogenetic analysis based on partial endoglucanase (egl) gene sequences (Poussier et al. 2000) (Figure S2) identified the isolates as R. solanacearum Phylotype IIB sequevar 51. The isolates were preserved in the COAD culture collection of the Universidade Federal de Vicosa, Brazil (codes COAD 2586, 2587 and 2588) and the corresponding egl DNA sequences were deposited in the GenBank (accession numbers MH350031, MH350032 and MH350033). Pathogenicity tests were conducted with all three isolates. Twenty microliters of bacterial suspensions (108 CFU/mL of saline solution) were injected at the base of the stem of eucalyptus (Eucalyptus urograndis), banana (Musa sp. cv. Maçã) and tomato (Solanum lycopersicum cv. Santa Clara) plants kept in a greenhouse (28 ± 2 °C) (Fonseca et al., 2016). Three plants of each host species were inoculated per bacterial isolate and plants injected with distilled water were used as controls. Necrotic symptoms appeared at the inoculation site on eucalyptus plants after 10 days. Wilt symptoms started at the top and progressed towards the base of the plants (Figure S1A). Tomato plants died within one week, and eucalyptus seedlings died after three weeks. The inoculated banana seedlings and all plants from the control treatments remained asymptomatic. Bacterial ooze was observed on freshly-cut transverse sections made on wilted eucalyptus seedlings (Figure S1B) and typical colonies of R. solanacearum were isolated from inoculated plants, fulfilling Koch's postulates. The correct diagnosis of the pathogen is the first step in the long-term process of developing effective management tools for this disease in Ecuador.

First Report of Bacterial Wilt Caused by Ralstonia solanacearum Phylotype IIB Sequevar 1 Affecting Tomato in Different Regions of Chile

First Report of Bacterial Wilt Caused by <i>Ralstonia solanacearum</i> Phylotype IIB Sequevar 1 Affecting Tomato in Different Regions of Chile

Sequencing, genome analysis and host range of a novel Ralstonia phage, RsoP1EGY, isolated in Egypt.

A novel Ralstonia phage was isolated from soil in Egypt. It was designated Ralstonia phage RsoP1EGY using our phage identifier naming approach to reflect the phage's bacterial host species, characteristics and origin. When tested, this phage specifically infected only race 3 biovar 2 phylotype IIB sequevar 1, and not non-race 3 biovar 2 strains of Ralstonia solanacearum. The phage has an icosahedral capsid of 60±5nm in diameter with a short tail of 15±5nm in length, typical of a podovirus. The genome of RsoP1EGY is 41,297bp in size, containing 50 open reading frames, with no significant sequence identity to any other reported R. solanacearum or non-Ralstonia phages, except to the recently deposited but unreported and unclassified Ralstonia phage DU_RP_I. RsoP1EGY is the first sequenced and characterized R. solanacearum phage isolated in Egypt.

PM 7/21 (2) Ralstonia solanacearum, R. pseudosolanacearum and R. syzygii (Ralstonia solanacearum species complex)

Specific scopeThis Standard describes a diagnostic protocol for Ralstonia solanacearum, Ralstonia pseudosolanacearum and Ralstonia syzygii, i.e. phylotype/sequevar strain in the Ralstonia solanacearum Species Complex (RSSC).It should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.Specific approval and amendmentApproved in 2003‐09. First revised in 2018‐02.

Molecular Epidemiology of Bacterial Wilt in the Madagascar Highlands Caused by Andean (Phylotype IIB-1) and African (Phylotype III) Brown Rot Strains of the Ralstonia solanacearum Species Complex.

The Ralstonia solanacearum species complex (RSSC) is a highly diverse cluster of bacterial strains found worldwide, many of which are destructive and cause bacterial wilt (BW) in a wide range of host plants. In 2009, potato production in Madagascar was dramatically affected by several BW epidemics. Controlling this disease is critical for Malagasy potato producers. The first important step toward control is the characterization of strains and their putative origins. The genetic diversity and population structure of the RSSC were investigated in the major potato production areas of the Highlands. A large collection of strains (n = 1224) was assigned to RSSC phylotypes based on multiplex polymerase chain reaction (PCR). Phylotypes I and III have been present in Madagascar for a long time but rarely associated with major potato BW outbreaks. The marked increase of BW prevalence was found associated with phylotype IIB sequevar 1 (IIB-1) strains (n = 879). This is the first report of phylotype IIB-1 strains in Madagascar. In addition to reference strains, epidemic IIB-1 strains (n = 255) were genotyped using the existing MultiLocus Variable-Number Tandem Repeat Analysis (MLVA) scheme RS2-MLVA9, producing 31 haplotypes separated into two related clonal complexes (CCs). One major CC included most of the worldwide haplotypes distributed across wide areas. A regional-scale investigation suggested that phylotype IIB-1 strains were introduced and massively spread via latently infected potato seed tubers. Additionally, the genetic structure of phylotype IIB-1 likely resulted from a bottleneck/founder effect. The population structure of phylotype III, described here for the first time in Madagascar, exhibited a different pattern. Phylotype III strains (n = 217) were genotyped using the highly discriminatory MLVA scheme RS3-MLVA16. High genetic diversity was uncovered, with 117 haplotypes grouped into 11 CCs. Malagasy phylotype III strains were highly differentiated from continental African strains, suggesting no recent migration from the continent. Overall, population structure of phylotype III involves individual small CCs that correlate to restricted geographic areas in Madagascar. The evidence suggests, if at all, that African phylotype III strains are not efficiently transmitted through latently infected potato seed tubers.

Prophage Rs551 and Its Repressor Gene orf14 Reduce Virulence and Increase Competitive Fitness of Its Ralstonia solanacearum Carrier Strain UW551.

We previously characterized a filamentous lysogenic bacteriophage, ϕRs551, isolated directly from the race 3 biovar 2 phylotype IIB sequevar 1 strain UW551 of Ralstonia solanacearum grown under normal culture conditions. The genome of ϕRs551 was identified with 100% identity in the deposited genomes of 11 race 3 biovar 2 phylotype IIB sequevar 1 strains of R. solanacearum, indicating evolutionary and biological importance, and ORF14 of ϕRs551 was annotated as a putative type-2 repressor. In this study, we determined the effect of the prophage and its ORF14 on the virulence and competitive fitness of its carrier strain UW551 by deleting the orf14 gene only (the UW551 orf14 mutant), and nine of the prophage’s 14 genes including orf14 and six out of seven structural genes (the UW551 prophage mutant), respectively, from the genome of UW551. The two mutants were increased in extracellular polysaccharide production, twitching motility, expression of targeted virulence and virulence regulatory genes (pilT, egl, pehC, hrPB, and phcA), and virulence, suggesting that the virulence of UW551 was negatively regulated by ϕRs551, at least partially through ORF14. Interestingly, we found that the wt ϕRs551-carrying strain UW551 of R. solanacearum significantly outcompeted the wt strain RUN302 which lacks the prophage in tomato plants co-inoculated with the two strains. When each of the two mutant strains was co-inoculated with RUN302, however, the mutants were significantly out-competed by RUN302 for the same colonization site. Our results suggest that ecologically, ϕRs551 may play an important role by regulating the virulence of and offering a competitive fitness advantage to its carrier bacterial strain for persistence of the bacterium in the environment, which in turn prolongs the symbiotic relationship between the phage ϕRs551 and the R. solanacearum strain UW551. Our study is the first toward a better understanding of the co-existence between a lysogenic phage and its carrier plant pathogenic bacterial strain by determining the effect of the prophage Rs551 and its repressor on the virulence and competitive fitness of its carrier strain UW551 of R. solanacearum.

Molecular and biological characterization of ϕRs551, a filamentous bacteriophage isolated from a race 3 biovar 2 strain of Ralstonia solanacearum.

A filamentous bacteriophage, designated ϕRs551, was isolated and purified from the quarantine and select agent phytopathogen Ralstonia solanacearum race 3 biovar 2 strain UW551 (phylotype IIB sequevar 1) grown under normal culture conditions. Electron microscopy suggested that ϕRs551 is a member of the family Inoviridae, and is about 1200 nm long and 7 nm wide. ϕRs551 has a genome of 7929 nucleotides containing 14 open reading frames, and is the first isolated virion that contains a resolvase (ORF13) and putative type-2 phage repressor (ORF14). Unlike other R. solanacearum phages isolated from soil, the genome sequence of ϕRs551 is not only 100% identical to its prophage sequence in the deposited genome of R. solanacearum strain UW551 from which the phage was isolated, but is also surprisingly found with 100% identity in the deposited genomes of 10 other phylotype II sequevar 1 strains of R. solanacearum. Furthermore, it is homologous to genome RS-09-161, resulting in the identification of a new prophage, designated RSM10, in a R. solanacearum strain from India. When ORF13 and a core attP site of ϕRs551 were either deleted individually or in combination, phage integration was not observed, suggesting that similar to other filamentous R. solanacearum ϕRSM phages, ϕRs551 relies on its resolvase and the core att sequence for site-directed integration into its susceptible R. solanacearum strain. The integration occurred four hours after phage infection. Infection of a susceptible R. solanacearum strain RUN302 by ϕRs551 resulted in less fluidal colonies and EPS production, and reduced motilities of the bacterium. Interestingly, infection of RUN302 by ϕRs551 also resulted in reduced virulence, rather than enhanced or loss of virulence caused by other ϕRSM phages. Study of bacteriophages of R. solanacearum would contribute to a better understanding of the phage-bacterium-environment interactions in order to develop integrated management strategies to combat R. solanacearum.

Interspecific Potato Breeding Lines Display Differential Colonization Patterns and Induced Defense Responses after Ralstonia solanacearum Infection.

Potato (Solanum tuberosum L.) is one of the main hosts of Ralstonia solanacearum, the causative agent of bacterial wilt. This plant pathogen bacteria produce asymptomatic latent infections that promote its global spread, hindering disease control. A potato breeding program is conducted in Uruguay based on the introgression of resistance from the wild native species S. commersonii Dun. Currently, several backcrosses were generated exploiting the high genetic variability of this wild species resulting in advanced interspecific breeding lines with different levels of bacterial wilt resistance. The overall aim of this work was to characterize the interaction of the improved potato germplasm with R. solanacearum. Potato clones with different responses to R. solanacearum were selected, and colonization, dissemination and multiplication patterns after infection were evaluated. A R. solanacearum strain belonging to the phylotype IIB-sequevar 1, with high aggressiveness on potato was genetically modified to constitutively generate fluorescence and luminescence from either the green fluorescence protein gene or lux operon. These reporter strains were used to allow a direct and precise visualization of fluorescent and luminescent cells in plant tissues by confocal microscopy and luminometry. Based on wilting scoring and detection of latent infections, the selected clones were classified as susceptible or tolerant, while no immune-like resistance response was identified. Typical wilting symptoms in susceptible plants were correlated with high concentrations of bacteria in roots and along the stems. Tolerant clones showed a colonization pattern restricted to roots and a limited number of xylem vessels only in the stem base. Results indicate that resistance in potato is achieved through restriction of bacterial invasion and multiplication inside plant tissues, particularly in stems. Tolerant plants were also characterized by induction of anatomical and biochemical changes after R. solanacearum infection, including hyperplasic activity of conductor tissue, tylose production, callose and lignin deposition, and accumulation of reactive oxygen species. This study highlights the potential of the identified tolerant interspecific potato clones as valuable genetic resources for potato-breeding programs and leads to a better understanding of resistance against R. solanacearum in potato.

Genetic and Pathogenic Diversity of Ralstonia solanacearum Causing Potato Brown Rot in China

Causing potato brown rot, Ralstonia solanacearum (R. solanacearum) strains are reported as one of the most destructive bacteria to potato (Solanum tuberosum L.) in China. In this study, 113 strains were isolated from potato, collected in the four major agroecological zones in China. The study showed that 102 strains belonged to the phylotype IIB sequevar 1 (race 3 biovar 2). The 11 remaining strains belonged to the phylotype I, sequevar 13, 17, 18, 16 or 14 M, a new sequevar closely related to sequevar 14. Thirty-four strains were further characterized according to their virulence at low temperature on three wild potato species. IIB-1 strains all belonged to high and moderate virulence, while others belonged to the low virulence group, which had limited pathogenicity.

Molecular characterization of Ralstonia solanacearum strains from Ethiopia and tracing potential source of bacterial wilt disease outbreak in seed potatoes

Bacterial wilt, caused by Ralstonia solanacearum, is emerging as a major threat to potato production in Ethiopia, reaching epidemic proportions in the Chencha district recently, with a prevalence of 97% of potato fields in 2015. The recent disease outbreak in the district coincided with a significant introduction of seed potatoes. This research was therefore initiated to genetically characterize the pathogen so as to trace its source, identify its relationship with outbreaks in the rest of the country, and make intervention recommendations. Ralstonia solanacearum isolates were sampled both from seed and ware potato fields in Chencha and from seed potato fields in production regions suspected of being potential sources of the pathogen. Multiplex PCR and phylogenetic analysis of partial endoglucanase gene sequences identified all of the isolates as phylotype IIB sequevar 1. VNTR sequence analysis distinguished 11 different haplotypes, nine of which were unique to the Chencha district. However, one of the haplotypes was common to all seed potato producer regions of Ethiopia except for the Shashemene area. The unique and diverse VNTR haplotypes of the pathogen in Chencha indicates that it is well established in the district. When a geographical map of the VNTR haplotypes was superimposed with the main cross‐regional seed potato distribution pattern of the country, it became evident that the pathogen was being disseminated via latently infected seed from the Holeta‐Jeldu area in the Central Highlands of Ethiopia. Identification of largely uninfected highland districts and multiplication of high‐grade seed potato exclusively in those districts should be given priority.

Phylogenetic relationships and population structure of Ralstonia solanacearum isolated from diverse origins in Taiwan

Bacterial wilt caused by Ralstonia solanacearum is a destructive disease for many crops. The aim of this study was to investigate the phylogenetic relationships and genetic structure of an R. solanacearum population from diverse origins in Taiwan. All 58 tested isolates belonged to phylotype I, except the two biovar 2 isolates from potato. These belonged to phylotype IIB sequevar 1 and were identical to known potato brown rot strains, which were probably introduced. Phylotype I isolates were grouped into 10 sequevars. Sequevar 15 was predominant (34 out of 56 isolates). Its distribution covered the whole island and it was largely associated with solanaceous crops such as tomato, and with tomato field soil. A total of 14 haplotypes were identified based on a partial endoglucanase gene sequence. Parsimony network analysis revealed that haplotype A was the oldest haplotype in the local population. It encompassed all but one of the sequevar 15 isolates. Large variation in virulence on tomato was observed among the 58 isolates, and seven pathotypes were identified. Significant genetic differentiation was detected among pathotypes. Moreover, genetic differentiation was detected between biovar 3 and biovar 4 subgroups and between the strains associated with solanaceous and non‐solanaceous species, but none was detected between strains from different geographic origins. The results suggest that the phylotype I population in Taiwan is homogeneous, while mutation and local adaptation to specific ecological niches keep shaping the population.

Comparative Effect of Low Temperature on Virulence and Twitching Motility of Ralstonia solanacearum Strains Present in Florida

Ralstonia solanacearum causes bacterial wilt on a wide range of plant hosts. Most strains of R. solanacearum are nonpathogenic below 20°C; however, Race 3 Biovar 2 (R3B2) strains are classified as quarantine pathogens because of their ability to infect crops, cause disease, and survive in temperate climates. We have identified race 1 biovar 1 Phylotype IIB Sequevar 4 strains present in Florida which were able to infect and produce wilt symptoms on potato and tomato at 18°C. Moreover they infected tomato plants at rates similar to strains belonging to R3B2. We determined that strains naturally nonpathogenic at 18°C were able to multiply, move in planta, and cause partial wilt when inoculated directly into the stem, suggesting that low temperature affects virulence of strains differently at early stages of infection. Bacterial growth in vitro was delayed at low temperatures, however it was not attenuated. Twitching motility observed on growing colonies was attenuated in nonpathogenic strains at 18°C, while not affected in the cool virulent ones. Using pilQ as a marker to evaluate the relative expression of the twitching activity of R. solanacearum strains, we confirmed that cool virulent strains maintained a similar level of pilQ expression at both temperatures, while in nonpathogenic strains pilQ was downregulated at 18°C.

Genetic diversity of Ralstonia solanacearum strains from China

A survey of bacterial wilt in China collected 286 strains of Ralstonia solanacearum from 17 plant species in 13 Chinese provinces to investigate genetic diversity using the biovar (bv.) and phylotype classification schemes. A phylotype-specific multiplex-PCR showed that 198 isolates belonged to phylotype I (bv. 3, 4 and 5) and 68 to phylotype II (bv. 2 and bv. 1). A phylogenetic analysis examined the partial sequence of the egl and hrpB gene of all strains and the genetic diversity of 95 representatives was reported, demonstrating that Chinese strains are partitioned into phylotype I (Asia) and II (Americas). Phylotype I strains (historically typed bv. 3, 4 and 5), had considerable phylogenetic diversity, including 10 different sequevars: seven previously described sequevars 12 to 18 and three new sequevars: 34, 44 and 48. Chinese strains Z1, Z2, Z3, Z7, Pe74 and Tm82 were not genetically distinguishable from the edible ginger reference strain ACH92 (r4-bv. 4) for sequevar 16. This is believed to be the first report of this ginger group in China. All Chinese bv. 2 strains falling into the genetically and phenotypically diverse phylotype II were placed into phylotype IIB sequevar 1 (historically the Andean race3-bv. 2 potato brown rot agent). In both the egl and hrpB sequence-based trees, strains isolated from mulberry were present in two distinct branches found in sequevars 12 and 48 (reference strains R292 and M2, respectively).